Effect of etoposide and amsacrine on mitotic progression of GM-130 and Hep-2 cell lines. The flow cytometry assay

It has been shown that inhibitors of topoisomerase II (topo II) etoposide and amsacrine results in accumulation of GM-130 and Hep-2 cells with 4c DNA amount. The differential analysis based on flow cytometry (Zenin et al., 2001) and enabled us to discriminate cells with 4c DNA--G2, M, including metaphase and anaphase cells and cells in pseudo-G1. 1 microM etoposide evoked cell accumulation in G2 phase, while 40 microM etoposide blocked cell proliferation, which was confirmed by a complete absence of both mitotic cells and 4c DNA cell accumulation. GM-130 and Hep-2 cells that were first blocked and then washed from nocodazole, and after that treated with 50 microM etoposide or 20 microM amsacrine, were shown to enter pseudo-G1 with 4c DNA amount per cell. In the presence of nocodazole, 4 and 40 microM amsacrine evoked transition of all mitotic cells to pseudo-G1 within 1 h. 15 or 30 minutes pulse treatments of GM-130 cells with 40 microM amsacrine in the presence of nocodazole, followed by incubation in drug-free medium, resulted in the similar transition of cells to pseudo-G1.     

Coordinated requirements of human topo II and cohesin for metaphase centromere alignment under Mad2-dependent spindle checkpoint surveillance

Cohesin maintains sister chromatid cohesion until its Rad21/Scc1/Mcd1 is cleaved by separase during anaphase. DNA topoisomerase II (topo II) maintains the proper topology of chromatid DNAs and is essential for chromosome segregation. Here we report direct observations of mitotic progression in individual HeLa cells after functional disruptions of hRad21, NIPBL, a loading factor for hRad21, and topo II alpha,beta by RNAi and a topo II inhibitor, ICRF-193. Mitosis is delayed in a Mad2-dependent manner after disruption of either or both cohesin and topo II. In hRad21 depletion, interphase pericentric architecture becomes aberrant, and anaphase is virtually permanently delayed as preseparated chromosomes are misaligned on the metaphase spindle. Topo II disruption perturbs centromere organization leading to intense Bub1, but no Mad2, on kinetochores and sustains a Mad2-dependent delay in anaphase onset with persisting securin. Thus topo II impinges upon centromere/kinetochore function. Disruption of topo II by RNAi or ICRF-193 overrides the mitotic delay induced by cohesin depletion: sister centromeres are aligned and anaphase spindle movements occur. The ensuing accumulation of catenations in preseparated sister chromatids may overcome the reduced tension arising from cohesin depletion, causing the override. Cohesin and topo II have distinct, yet coordinated functions in metaphase alignment.     

A Topoisomerase II-Dependent Checkpoint in G2-Phase Plant Cells Can Be Bypassed by Ectopic Expression of Mitotic Cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclin-dependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.

Key Words:

Plant cyclin B2, Topoisomerase II, ICRF-193, G2 checkpoint, Microtubules     

A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed by ectopic expression of mitotic cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.     

Revised genetic requirements for the decatenation G2 checkpoint: The role of ATM

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.     

Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase

DNA damage by double-strand breaks induces arrest during interphase in mammalian cells. It is not clear whether DNA damage can arrest cells in mitosis. We show here that three human cell lines, HeLa, U2OS, and HCT116, do not delay in mitosis in response to double-strand breaks induced during mitosis by gamma irradiation or by adriamycin. Durable arrest at metaphase occurs, however, with ICRF-193, a topoisomerase II inhibitor that does not damage DNA. Arrest with ICRF-193 is not accompanied by recruitment of Mad2 or Bub1 to kinetochores, nor by phosphorylation of the histone H2AX, indicating arrest by ICRF-193 is not due to activation of the spindle assembly checkpoint, nor is it a response to DNA damage. VP-16, another decatenation inhibitor, induces metaphase arrest only at concentrations well above those that induce DNA damage. We conclude that decatenation failure, but not DNA damage, creates metaphase arrest in mammalian cells.     

DNA catenations that link sister chromatids until the onset of anaphase are maintained by a checkpoint mechanism

Treatment of Allium cepa meristematic cells in metaphase with the topoisomerase II inhibitor ICRF-193, results in bridging of the sister chromatids at anaphase. Separation of the sisters in experimentally generated acentric chromosomal fragments was also inhibited by ICRF-193, indicating that some non-centromeric catenations also persist in metaphase chromosomes. Thus, catenations must be resolved by DNA topoisomerase II at the metaphase-to-anaphase transition to allow segregation of sisters. A passive mechanism could maintain catenations holding sisters until the onset of anaphase. At this point the opposite tension exerted on sister chromatids could render the decatenation reaction physically more favorable than catenation. But this possibility was dismissed as acentric chromosome fragments were able to separate their sister chromatids at anaphase. A timing mechanism (a common trigger for two processes taking different times to be completed) could passively couple the resolution of the last remaining catenations to the moment of anaphase onset. This possibility was also discarded as cells arrested in metaphase with microtubule-destabilising drugs still displayed anaphase bridges when released in the presence of ICRF-193. It is possible that a checkpoint mechanism prevents the release of the last catenations linking sisters until the onset of anaphase. To test whether cells are competent to fully resolve catenations before anaphase onset, we generated multinucleate plant cells. In this system, the nuclei within a single multinucleate cell displayed differences in chromosome condensation at metaphase, but initiated anaphase synchronously. When multinucleates were treated with ICRF-193 at the metaphase-toanaphase transition, tangled and untangled anaphases were observed within the same cell. This can only occur if cells are competent to disentangle sister chromatids before the onset of anaphase, but are prevented from doing so by a checkpoint mechanism.     

Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events

ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909-4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193. Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes.     

DNA signals for G2 checkpoint response in diploid human fibroblasts

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included γ-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4–12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.     

Topoisomerase II-DNA complexes trapped by ICRF-193 perturb chromatin structure

DNA topoisomerase II (topo II) is involved in unlinking replicating DNA and organizing mitotic chromosomes. Topo II is the target of many antitumour drugs. Topo II inhibition results in extensive catenation of newly replicated DNA and may potentially perturb chromatin assembly. Here, we show that the topo II inhibitor ICRF-193 does not prevent the bulk of nucleosome deposition, but perturbs nucleosome spacing in Xenopus egg extracts. This is due to the trapping of topo II-closed clamps on the DNA rather than increased DNA catenation. Inhibition of replicative DNA decatenation has in itself little or no effect on nucleosome deposition and spacing, suggesting that DNA can easily accommodate the sharp bending constraints imposed by the co-habitation of nucleosomes and catenane nodes. Chromatin perturbation by topo II clamps may explain some dominant cellular effects of ICRF-193. Nucleosome-driven bending of precatenane nodes may facilitate their unlinking by topo II during unperturbed replication.     

Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1

Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving gamma-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.     

Topoisomerase II and histone deacetylase inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway

When early prophase PtK(1) or Indian muntjac cells are exposed to topoisomerase II (topo II) inhibitors that induce little if any DNA damage, they are delayed from entering mitosis. We show that this delay is overridden by inhibiting the p38, but not the ATM, kinase. Treating early prophase cells with hyperosmotic medium or a histone deacetylase inhibitor similarly delays entry into mitosis, and this delay can also be prevented by inhibiting p38. Together, these results reveal that agents or stresses that induce global changes in chromatin topology during G2 delay entry into mitosis, independent of the ATM-mediated DNA damage checkpoint, by activating the p38 MAPK checkpoint. The presence of this pathway obviates the necessity of postulating the existence of multiple "chromatin modification" checkpoints during G2. Lastly, cells that enter mitosis in the presence of topo II inhibitors form metaphase spindles that are delayed in entering anaphase via the spindle assembly, and not the p38, checkpoint.     

ATR enforces the topoisomerase II-dependent G2 checkpoint through inhibition of Plk1 kinase

An ATR-dependent G(2) checkpoint responds to inhibition of topoisomerase II and delays entry into mitosis by sustaining nuclear exclusion of cyclin B1-Cdk1 complexes. Here we report that induction of this checkpoint with ICRF-193, a topoisomerase II catalytic inhibitor that does not cause DNA damage, was associated with an ATR-dependent inhibition of polo-like kinase 1 (Plk1) kinase activity and a decrease in cyclin B1 phosphorylation. Expression of constitutively active Plk1 but not wild type Plk1 reversed ICRF-193-induced mitotic delay in HeLa cells, suggesting that Plk1 kinase activity is important for the checkpoint response to ICRF-193. G(2)/M synchronized normal human fibroblasts, when treated with ICRF-193, showed a decrease in cyclin B1 phosphorylation and Plk1 kinase activity despite high cyclin B1-Cdk1 kinase activity. G(2) fibroblasts that were treated with caffeine to override the checkpoint response to ICRF-193 displayed a high incidence of chromosomal aberrations. Taken together, these results suggest that ATR-dependent inhibition of Plk1 kinase activity may be one mechanism to regulate cyclin B1 phosphorylation and sustain nuclear exclusion during the G(2) checkpoint response to topoisomerase II inhibition. Moreover, the results demonstrate an important role for the topoisomerase II-dependent G(2) checkpoint in the preservation of human genomic stability.     

Mitotic Bypass Via An Occult Cell Cycle Phase Following DNA Topoisomerase II Inhibition In p53 Functional Human Tumor Cells

Cell cycle checkpoints guard against the inappropriate commitment to critical cell events such as mitosis. The bisdioxopiperazine ICRF-193, a catalytic inhibitor of DNA topoisomerase II, causes a reversible stalling of the exit of cells from G2 at the decatenation checkpoint (DC) and can generate tetraploidy via the compromising of chromosome segregation and mitotic failure. We have addressed an alternative origin – endocycle entry - for the tetraploidisation step in ICRF-193 exposed cells. Here we show that DC-proficient p53-functional tumour cells can undergo a transition to tetraploidy and subsequent aneuploidy via an initial bypass of mitosis and the mitotic spindle checkpoint. DC-deficient SV40-tranformed cells move exclusively through mitosis to tetraploidy. In p53-functional tumour cells, escape through mitosis is enhanced by dominant negative p53 co-expression. The mitotic bypass transition phase (termed G2endo) disconnects cyclin B1 degradation from nuclear envelope breakdown and allows cells to evade the action of Taxol. G2endo constitutes a novel and alternative cell cycle phase - lasting some 8 h - with distinct molecular motifs at its boundaries for G2 exit and subsequent entry into a delayed G1 tetraploid state. The results challenge the paradigm that checkpoint breaching leads directly to abnormal ploidy states via mitosis alone. We further propose that the induction of bypass could: facilitate the covert development of tetraploidy in p53 functional cancers, lead to a misinterpretation of phase allocation during cell cycle arrest and contribute to tumour cell drug resistance.     

Chromosomes with Two Intact Axial Cores Are Induced by G2 Checkpoint Override: Evidence That DNA Decatenation Is not Required to Template the Chromosome Structure

Here we report that DNA decatenation is not a physical requirement for the formation of mammalian chromosomes containing a two-armed chromosome scaffold. 2-aminopurine override of G₂ arrest imposed by VM-26 or ICRF-193, which inhibit topoisomerase II (topo II)–dependent DNA decatenation, results in the activation of p34^cdc2 kinase and entry into mitosis. After override of a VM-26–dependent checkpoint, morphologically normal compact chromosomes form with paired axial cores containing topo II and ScII. Despite its capacity to form chromosomes of normal appearance, the chromatin remains covalently complexed with topo II at continuous levels during G₂ arrest with VM-26. Override of an ICRF-193 block, which inhibits topo II–dependent decatenation at an earlier step than VM-26, also generates chromosomes with two distinct, but elongated, parallel arms containing topo II and ScII. These data demonstrate that DNA decatenation is required to pass a G₂ checkpoint, but not to restructure chromatin for chromosome formation. We propose that the chromosome core structure is templated during interphase, before DNA decatenation, and that condensation of the two-armed chromosome scaffold can therefore occur independently of the formation of two intact and separate DNA helices.     

Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage

A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4-/- and KU70-/- cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54-/- cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.     

Degradation of ATM-Independent Decatenation Checkpoint Function in Human Cells is Secondary to Inactivation of p53 and Correlated with Chromosomal Destabilization

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.

Key Words:

Checkpoints, DNA damage, Decatenation, Topoisomerase II, ICRF-193, Radiation     

Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.     

Induction of fusion-competent myoblast-specific gene expression during myogenic differentiation of Drosophila Schneider cells by DNA double-strand breaks or replication inhibition

Differentiation of Drosophila Schneider cells caused by DNA double-strand break (DSB)-inducing topoisomerase II (topo II) inhibitors were attenuated by ICRF-193, a non-DNA-damaging topo II inhibitor. ICRF-193 did not inhibit differentiation induced by neocarzinostatin (NCS), a drug that causes DNA DSBs independent of topo II. Schneider cells differentiated upon treatment with gamma-ray. These results suggest that DNA DSBs induce myogenic differentiation of Schneider cells. We also found DNA replication inhibitors, hydroxyurea (HU), aphidicolin, and ethylmethanesulfonate (EMS) induced myogenic differentiation of Schneider cells. HU-induced differentiation was inhibited upon pretreatment of cells with chemical inhibitors of PP 1/2A, p38 MAPK, JNK, and proteasome. RT-PCR analysis revealed that the expressions of fusion-competent myoblast-specific genes lmd, sns, and del were induced in Schneider cells upon treatment with NCS or HU, whereas expressions of three founder cell-specific genes, duf, ants, and rols, were undetectable. These results indicate that the expression of fusion competent-myoblast-specific genes is induced during myogenic differentiation of Drosophila Schneider cells by DNA DSBs or replication inhibition.